Motor function depends on specification of hundreds of distinct motor neuron (MN) subtypes within the developing neural tube. The motor neuron subtype identity is determined by anteroposterior (AP) patterning signals that subdivide the otherwise uniform neural tube into molecularly and functionally distinct segments. In comparison to the detailed understanding of dorsoventral patterning and specification of the generic MN identity, relatively little is known about the molecular mechanisms underlying anteroposterior patterning of the spinal cord and acquisition of MN subtype identity. Here we take advantage of our ability to direct differentiation of ES cells into motor neurons acquiring distinct subtype identities in vitro. We will use this system to study the genetic cascade underlying the acquisition of anteroposterior identity: 1) we will define extrinsic signals that regulate AP patterning;2) we will define the time point during which AP identity is consolidated and 3) we will define molecular programs that underlie this process. Gaining insight into the process of acquisition of specific neuronal segmental and subtype identities during ES cell differentiation will have an impact on the utilization of ES cells to study and treat neurodegenerative diseases. The differential susceptibility of individual motor neuron subtypes to degeneration might reveal new potential targets for therapeutic intervention. In addition, our ability to direct MN subtype identity will be essential for effective implementation of MN cell replacement therapies.